The goal of this project is to understand the mechanisms involved in the killing of bacteria and other microbial invaders by human neutrophils and related cells. 2 molecular entities of central importance, the enzyme, NADPH oxidase and voltage-gated proton channels are the focus of this project. NADPH oxidase produces superoxide anion, the precursor to reactive oxygen species that kill microbes. This enzyme works by transporting electrons across the cell membrane, which generates an electrical current that will be measured using the "perforated-patch" and excised-patch voltage-clamp techniques. As demonstrated recently, (DeCoursey et al, 2003) the depolarization caused by this electron current would stop enzyme function almost immediately, if there were no charge compensation mechanism. As hypothesized, (Hendeson et al, 1987) charge compensation is achieved by proton flux through channels. Proton channel-mediated currents will be studied using the perforated-patch technique. This approach, which allows simultaneous study of both NADPH oxidase and proton channel function in living, responsive cells, is currently used only by our lab. (1) We will determine the voltage-, temperature-, and substrate-dependence of NADPH oxidase. (2) We will evaluate the proposed role of proton channels in charge compensation, by studying superoxide production in phagocytes (neutrophils and eosinophils), inhibiting proton channels with Zn2+, and using ionophores. We will examine a recent proposal that K channels play important roles in charge compensation and microbe killing (Ahluwalia et al, 2004). (3) We will determine the dependence of NADPH oxidase function on pHo and pHi. In our hypothesis, 1 mechanism of communication between NADPH oxidase and proton channels is intracellular pH. (4) The coordinated regulation of NADPH oxidase and proton channels will be explored. Several physiological NADPH oxidase agonists will be studied to determine their effects on both the oxidase and proton channels. We will inhibit signaling pathways to determine how proton channel function is regulated. Human phagocytes manage to turn on NADPH oxidase and proton channels when they kill bacteria - this project will clarify how the coordinated regulation of these 2 key molecules is accomplished. (5) We will identify the proton channel molecule and gene.